Primer sets for analyzing cytochrome P450 isoenzymes expression

ABSTRACT

Hepatocyte culturing system, primer sets and an analytical method for selectively detecting and quantitatively assessing the levels of mRNA expression of the major isoenzymes of cytochrome P450 (CYP450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2 and 4A1), fatty acyl-CoA oxidase (FACO) and select Phase II conjugating enzymes (UDPGT, GST and ST) in the rat using specific 5&#39; and 3&#39; oligonucleotide primers and reverse transcriptase-polymerase chain reaction. The method closely reproduces the expression obtained from rat liver tissue following treatment with the same enzyme inducers. Constitutive and inducible expression was maintained by resuspending, culturing and then overlaying adult rat hepatocytes with an extracellular matrix such as Matrigel®.

This application is a continuation in part of Ser. No. 08/645,067, nowabandoned, filed May 13, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of detecting expression of isoenzymesof cytochrome P450 (CYP450) and Phase II conjugating enzymes in the rat.More specifically, this invention relates to specific 5' and 3' specificoligonucleotide primers, as well as a method of using the same withreverse transcriptase-polymerase chain reaction (RT-PCR) to detect mRNAexpression of the major isoenzymes of CYP450 and fatty acyl-CoA oxidasein the rat. This invention includes the development of an in vitroculture system using rat hepatocytes which has been optimized forexpression of both cytochrome P450 and Phase II conjugating enzymes.

2. Description of the Prior Art

The cytochrome P450 mixed function oxidases (MFO) are a group of enzymeswhich are predominantly expressed within the liver, kidney, lung andintestine of mammalian species where they play an important role in theoxidative metabolism of both endogenous and exogenous (xenobiotic)compounds. The role of the various members of this enzyme superfamily inthe metabolism of drugs and chemicals, as well as their potential rolein the generation of toxic metabolites and chemical-inducedcarcinogenesis, is well established.

In particular, induction of specific CYP450 isoforms has been associatedwith drug-drug interactions in humans and increases in liver weight,proliferation of the endoplasmic reticulum and non-genotoxic livercarcinogenicity and tumorigenicity in rodents. Guengerich, Cancer Res48:2946-2954, 1988; Lake, Ann Rev Phaacol Toxical 35: 483-507, 1995;Hankinson, Ann Rev Pharmacol Toxicol 35:307-340, 1995; Parkinson,Toxicol Pathol 24:45-57, 1996; and Kirby et al., Toxicol Pathol24:458-467, 1996; and Burchell et al., Pharmacol Ther 43:261-289, 1989.

In addition to CYP450 enzymes, Phase II enzymes, which are involved inthe conjugation of xenobiotics and their metabolites for excretion, havealso been associated with the bioactivation of xenobiotics to toxic andtumorigenic metabolites. Parkinson, Casarett & Doull's Toxicology, TheBasic Science of Poisons (Ed., Klaasen CD), 5th Edn, pp 113-186,McGraw-Hill, Inc., New York, 1996; Boberg, Cancer Res. 43:5163-5173,1983; Curran et al., Endocrine Rev 12:135-150, 1991; Bock,Pharmacogenetics 4:209-218, 1994; and Monks, et al., Toxicol ApplPharmacol 106:1-19, 1990.

Examples of these Phase II enzymes include uridinediphosphate-glucuronosyltransferases (UDPGT), glutathione-S-transferases(GST), and sulfotransferases (ST). Historically, in vivo models arecommonly used for the study of chemical-induced enzyme expression andhepatotoxicity. However, this approach is costly, time consuming andrequires large quantities of test material.

Several approaches have been used to monitor the regulation ofcytochrome P450 (CYP450) enzymes following exposure to xenobiotics. Theapproach most often employed is to measure the enzymatic profiles ofmicrosomal protein fractions using enzyme selective substrates. Althoughthis technique is useful for the study of substrate specificities,enzyme kinetics, and metabolism of chemicals there are severaldisadvantages which can limit the application of this technique inassessing the biochemical regulation of these enzymes. Two importantdisadvantages are that CYP450 enzyme activity requires additionalcofactors (e.g., requirement for the presence of heme) and can shownon-selectivity for, or be inhibited by certain chemical substrates(e.g., ketoconazole and metyrapone), resulting in potentially misleadingor inaccurate assessments of enzyme activity.

Today, the chemical industry (Pharmaceutical and Chemical manufacturers)recognize the value in developing in vitro techniques to assess thesafety and efficacy of drugs and chemicals at an early stage ofdevelopment. In vitro techniques most commonly used to study theexpression of hepatic metabolizing enzymes and cytotoxicity includeprecision-cut liver slices (Brendel et al., Methods in Toxicology (Ed.Tyson and Frazier), Vol 1 A. pp 222-243, Academic Press Inc. NY., 1993;and Gandolfi et al., Toxicol. Pathol. 24:58-61, 1996), primary culturesof hepatocytes and immortalized cell lines. Donato et al., In Vitro CellDevelop Biol 30A:574-580, 1994; and MacDonald et al., Human and ExpToxicol 13:439-444, 1994.

However, without exception, these in vitro systems have limitations intheir applications. For example, continuously dividing cell lines failto preserve their ability to express or induce specific Phase I and IImetabolizing enzymes, resulting in enzymatic activities which are eitherabsent or too low to be measured. In addition, a loss of more than 50%of the total metabolizing enzyme levels have been reported within 24 hrof culture using non-dividing whole cell systems (e.g., precision-cuttissue slices and primary cell culture systems). Guzelian et al., DrugMetabol Rev 10:793-809, 1989; Waxman et al., Biochem J. 271:113-119,1990; Paine, Chem Biol Interac 747:1-31, 1990; and Dunn et al.,Biotechnol Prog 7:237-245, 1991.

A number of reports have demonstrated that primary hepatocytes culturedunder conditions which restore normal cell's morphology and liverspecific gene expression, can respond to xenobiotics with induction ofspecifically inducible CYP450 enzymes to levels comparable with thoseachieved in vivo. Bissel et al., Ann NY Acad Sci 349:85-98, 1980; Isomet al., J Cell Biol 105:2877-2885, 1987; Ben-Ze'ev et al., Proc NatlAcad Sci 85:2161-2165, 1988; Schuetz et al., J Cell Physiol 134:309-323,1988; Musat et al., Hepatology 18:198-205, 1993; Arterburn et al.,Hepatology 21:175-187, 1995; Kocarek et al., Mol Pharmacol 43:328-334,1992; Sidhu et al., In Vitro Toxicol 7:225-242, 1994; and Zurlo et al.,In Vitro Cell Develop Biol 32:211-220, 1996.

Examples of these cell culture conditions include the use of anextracellular matrix (ECM), Schuetz et al., J Cell Physiol 134:309-323,1988, chemically defined culture media conditions and hepatocytesco-cultured with non-parenchymal cells. Begue et al., Hepatol 4:839-842,1984; Rogiers et al., Biochem Pharmacol 40:1701-1706, 1990; Donato etal., In vitro Cell Develop Biol 30A:825-832, 1994; Guzelian et al., ProcNatl Acad Sci 85:9783-9787, 1988; Kocarek et al., Mol Pharmacol38:440-444, 1990; Kocarek, In Vitro Cell Develop Biol 29A:62-66, 1992;and Kocarek et al., Biochem Pharmacol 48:1815-1822, 1994.

More recently, polyclonal and monoclonal antibodies have been generatedagainst various isoenzymes of CYP450 found in rat, thereby allowing fora more selective and defined analysis of CYP450 expression, Parkinson etal., Meth. Enzymol. 206: 233-245, 1991. This approach to monitoringchanges in CYP450 isoenzymes has many advantages over those whichmonitor enzyme activity, particularly with regard to enzymes which areregulated post-translationally (e.g., CYP2E1). However, the success ofthis approach is dependent on the quality and availability of reagents(e.g., polyclonal versus monoclonal antibodies) and may lack thespecificity for determining enzyme subtype expression (e.g., CYP3A1 andCYP3A2). Moreover, the generation of antibodies and the measurement ofproteins using Western immunoblot analysis are both labor intensive andtime consuming and cannot be readily implemented when new enzymes areidentified.

A number of reports have described use of ECM systems in studying theexpression of liver-specific gene regulation in primary rat hepatocytes,Kocarek et al., Drug Metabol Dispos 23:415-421, 1995, Waxman et al.,Biochem J 271:113-119, 1990, Sidhu et al., In Vitro Toxicol 7:225-242,1994, and Zurlo et al., In Vitro Cell Develop Biol 32:211-220, 1996.

These reports indicate that 1) hepatocyte genes expression decreasesmarkedly after cell isolation, 2) hepatocyte isolation procedures resultin altered expression of CYP450 mRNAs, 3) hepatocyte gene expression isrestored after several days in culture, 4) the maintenance and/orinducibility of one or more P450 isoenzymes are lost during the first 48hr in culture, and 5) hepatocytes cultured on extracellularmatrix-coated plates, and under specific culture conditions (e.g.,modified Chee's medium) maintain a wide range of CYP450 isoenzymeexpression and the activities of certain CYP450 enzymes are enhancedfollowing exposure to CYP450 inducing agents.

Accordingly, the present inventors sought to develop a feasible,reproducible and simple in vitro system for evaluating xenobiotics asCYP450 enzyme inducers, which could be used for routine toxicologyapplications. The inventors have optimized the ECM system to study andmonitor a broad range of liver biotransformation enzymes at the mRNA andprotein levels. In contrast with the classical methods in whichhepatocytes are seeded on ECM-coated dishes (e.g., collagen orMatrigel®), and then maintained on Matrigel® (sandwich or overlay), thepresent invention optimizes rat hepatocyte culture conditions for enzymeexpression by suspending the hepatocytes in Matrigel® before seedingthem in culture. Rat hepatocytes cultured under these culture conditionsshow an enhanced level of expression of a variety of liver metabolizingenzymes within 24 hr after initial plating in response to chemicalinducers. This permits induction and maintenance in culture ofliver-specific biotransformation enzyme genes for greater than sevendays at both the mRNA and protein levels. More importantly, rathepatocytes responded very similar to adult rat liver when exposedrepeatedly to the same or similar types of inducing agents.

The inventors have demonstrated this improved rat hepatocyte culturesystem by utilizing prototypical CYP450 enzyme inducers, including PB asan inducer of the CYP2B subfamily, HC as an inducer of the CYP3Asubfamily, 3MC as an inducer of the CYPLA subfamily and CLO as aninducer of the CYP4A subfamily (and FACO). In all of the in vitroexperiments conducted, substantially complete agreement in the detectionof CYP450 gene expression was found between the RT-PCR and westernimmunoblotting techniques.

These results show that CYP450 enzyme expression is regulated primarilyat the mRNA level. Nebert, Biochem Pharmacol 47:25-37, 1994, Gonzalez etal., Pharmacogenetics 3:51-57, 1993 and Gonzalez et al., FASEB J10:1112-1117, 1996. This is particularly true of the inducible forms theCYP450 enzymes, including CYP1A1/2, CYP2B1, CYP3A1/2 and CYP4A1. Theseenzymes are shown to be constitutively expressed at low or undetectablelevels in this in vitro culture system but are markedly increased uponexposure to CYP450 inducing agents at both the mRNA and protein levels.The induction of these CYP450 enzymes involves transcriptionalactivation of the CYP450 genes, which may also involve messagestabilization, resulting in an increase in the levels of mRNA and newlysynthesized protein.

The expression of CYP2E1 mRNA, which is constitutively expressed invitro at both the mRNA and protein levels, is known to be increasedgenerally by protein stabilization, although mRNA stabilization and/orincreased efficiency of mRNA translation may also be involved. Hunt etal., Xenobiotica 21:1621-1631, 1991 and Raucy et al., Critical RevToxicol 23:1-20, 1993.

CYP2C11, an adult male rat-specific CYP450 isoform, has been shown to beconstitutively expressed and noninducible in male rat liver. Waxman etal., Biochem 88:6868-6871, 1991, Morohashi et al., FASEB J 10:1569-1577,1996 and Prough et al., FASEB J 10:1369-1377, 1996. This enzyme has beenshown to be partially regulated by androgenic hormones in vivo and thedisruption of circulating levels of growth hormone patterns byxenobiotics can decrease expression of this enzyme.

As expected, CYP2C11 was found to be constitutively expressed at lowlevels and noninducible by the CYP450 inducing agents in male rathepatocytes cultured under the present conditions.

In agreement with other investigators, we have observed that maintenanceof 3MC-mediated induction of the CYP1A1 and CYP1A2 enzymes, PB-mediatedinduction of the CYP2B11 and CYP3A1 enzymes, HC-mediated induction ofCYP3A1, CYP3A2, and CYP2B enzymes, and CLO-mediated induction of theCYP4A1 and FACO enzymes in rat hepatocytes, is critically dependent onthe presence of an ECM (e.g., Matrigel®). Waxman et al., Biochem J271:113-119, 1990, Jauregui et al., Xenobiotica 21:1091-1106, 1991, andKocarek et al., Mol Pharmacal 43:328-334, 1992.

We have also observed that CYP3A1 and CYP3A2 are induced by both steroidand PB-type inducers and the induction is dependent on the concentrationof the inducer in the medium. For example, treatment of hepatocytecultures with hydrocortisone, at concentrations as low as 10 μM, resultsin the induction of the CYP3A1 and CYP3A2 and CYP2B1 enzymes at mRNAlevel, whereas lower doses (e.g, 0.1 μM) did not affect enzymeexpression. Like CYP3A1, CYP2B1 mRNA levels increased in a dose-relatedmanner in the presence of HC.

Another significant finding was the effect of CLO on hepatocytescultured on Matrigel®. We have found, in agreement with in vivo studies,that CLO increased the expression of the CYP4A1, CYP2E1 and FACO enzymesat the mRNA level. Tugwood et al., EMBO J 11:433-439, 1992, Gulick etal., Proc Natl Acad Sci 91:11012-11016, 1994 and Johnson et al., FASEB J10:1241-1248, 1996.

This finding suggests that activation of peroxisomal enzymes can occurin this system, that CLO causes an increase in the expression ofmultiple CYP450 enzymes and that CLO can up-regulate the expression ofthe CYP2E1 enzyme at the mRNA level. The effect of CLO on CYP2E1expression has been observed following in vivo exposures.

Phase II enzymes are of considerable importance in toxicology. Thepharmacological and toxicological effect of many reactive endogenous andexogenous compounds depends on their rate of formation and elimination,which involves Phase II conjugating enzymes such as UDPGT, GST and ST.The absence or presence of these enzymes has been associate withcarcinogenicity and adverse reactions of certain drugs and chemicals.

To further characterize our in vitro system, we have studied the effectsof liver enzyme inducers on the expression of UDPGT, GST-Ya and ST atthe mRNA level. We have found that these enzymes are constitutivelyexpressed and induced by prototypical CYP450 inducers, similar to theinducible CYP450 isoforms.

In contrast with CYP450 enzymes, Phase II enzyme expression has beenscarcely studied in cell culture. However, several reports have shownthat Phase II conjugating enzymes can be maintained in culture usingmodified culture conditions (Grant et al., Biochem Pharmacol35:2979-2982, 1986, Vandenberghe et al., In vitro Cell Develop Biol24:281-288, 1988 and Vandenberghe et al., Biochem Pharmacol37:2481-2485, 1988), ECM (Kane et al., In vitro Cell Develop Biol27A:953-960, 1996, Judah et al., Toxic Appl Pharmacol 125:27-33, 1994)or co-culture systems. Rogiers et al., Biochem Pharmacol 40:1701-1706,1990.

We have found that these enzymes can be maintained and induced inhepatocytes cultured with Matrigel®. We have found that phenobarbital,as well as hydrocortisone, markedly enhance the expression of theseenzymes. The regulation of these Phase II conjugating enzymes byglucocorticoids has been previously demonstrated in cultured rathepatocytes. The data presented here support the potential utility ofthis culture system for assessing the induction of Phase II conjugatingenzymes in the rat by new chemical entities (NCE).

SUMMARY OF THE INVENTION

An object of the present invention is to selectively assay CYP450expression in both in vivo (whole animal) and in vitro (hepatocyte orother expression system) systems.

Another object of the present invention is to assay CYP450 expressionwithout regard to enzyme subtype.

A still other object of the present invention is to provide an in vitrorat hepatocyte culturing system (and analytical method) for determiningthe effects of xenobiotics on the expression of adult rat liver CYP450and Phase II conjugating enzymes.

These objects and others are provided by the present invention, which isa method of using reverse transcriptase-polymerase chain reaction(RT-PCR) with particular PCR primers to detect and quantitate the levelsof mRNA expression of the major isoenzymes of CYP450, fatty acyl-CoAoxidase (FACO) and select Phase II conjugating enzymes in both in vivoand in vitro systems. This invention also relates to the specificoligonucleotide 5' and 3' PCR primers which can be used to amplify DNAencoding selected CYP450 isoenzymes, FACO and select Phase IIconjugating enzymes, as well as kits containing sets of the primer. Thisinvention also includes the development of an in vitro rat hepatocyteculturing system which has been optimized for use in determiningchemical-mediated induction of CYP450 and Phase II conjugating enzymes.

As a result of the present invention, expression of CYP450 enzymes canbe efficiently and selectively measured at the mRNA level in any tissue,especially following exposure to drugs and chemicals. This technique isextremely sensitive, quantitative and can be automated for diagnosticapplications. This cell culture system and analytical methodology isalso applicable to routine toxicology screening for the assessment ofchemically-induced changes in liver CYP450 enzyme expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b illustrate the results obtained using RT-PCR to assessthe levels of CYP450 FACO and Phase II mRNA expression in the livers ofmale rats exposed to various chemical inducers of CYP450 in in vivo andin vitro, respectively.

FIGS. 2a and 2b illustrate the results obtained using Westernimmunoblotting to assess the levels of CYP450 apoprotein in the liversof male rats exposed to various chemical inducers of CYP450 in in vivoand in vitro, respectively.

FIGS. 3a-3z illustrate the results obtained using RT-PCR, westernImmunoblotting and substrate-specific enzyme activities to assess thelevels of CYP450, FACO and Phase II enzyme expression in the livers ofmale rats exposed to various chemical inducers.

DETAILED DESCRIPTION OF THE INVENTION

This invention is a method of detecting levels of mRNA expression ofisoenzymes of cytochrome P450 mixed function oxidases (CYP450 MFOs)using reverse transcriptase-polymerase chain reaction and specificoligonucleotide PCR primers. The CYP450 MFOs present one of the body'smost important mechanisms of defense against chemical-induced toxicity.This superfamily of catabolic enzymes oxidize both endogenous andexogenous (xenobiotic) compounds, converting them to hydrophilicmetabolites which can be readily removed from the body.

In some instances however, metabolism of chemicals by the CYP450 enzymesmay be undesirable or detrimental to the body. For instance, metabolismof certain compounds (e.g., acetaminophen) can lead to toxic or reactiveintermediates, resulting in target organ toxicity and/or carcinogenicinsult.

Furthermore, with chemical-based therapeutics, induction of CYP450isoenzymes can lead to metabolism of and/or accelerated removal ofdrugs, which may limit their desired pharmacological actions. Thisphenomena, known as "autoinduction", can be problematic in drugdiscovery and an obstacle to the development of adequate therapeutics.Finally, species differences in metabolic capabilities of therapeuticcandidates may suggest that responses in certain animal models are notrelevant for man. Therefore, it is extremely important to monitor theexpression of CYP450 isoenzymes in the early stages of drug discovery,to assist in the development of safe and effective therapeutics.

The level of expression of CYP450 isoenzyme and fatty acyl CoA oxidaseis determined by quantitating amplified cDNA (mRNA) obtained through theuse of oligonucleotide primer sets that specifically amplify for theindividual isoenzyme or oxidase.

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 1A1 contains oligonucleotides with thesequences

    (5') CTGGTTCTGGATACCCAGCTG (3')                                                                      (SEQ ID NO:1)                                             - (5') CCTAGGGTTGGTTACCAGG (3')      (SEQ ID NO:2).                    

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 1A2 contains oligonucleotides with thesequences

    (5') GTCACCTCAGGGAATGCTGTG (3')                                                                      (SEQ ID NO:3)                                             - (5') GTTGACAATCTTCTCCTGAGG (3')    (SEQ ID NO:4).                    

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 2B1/2 contains oligonucleotides with thesequences

    (5') GAGTTCTTCTCTGGGTTCCTG (3')                                                                      (SEQ ID NO:5)                                             - (5') ACTGTGGGTCATGGAGAGCTG (3')    (SEQ ID NO:6).                    

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 2C11 contains oligonucleotides with thesequences

    (5') CTGCTGCTGCTGAAACACGTG (3')                                                                      (SEQ ID NO:7)                                             - (5') GGATGACAGCGATACTATCAC (3')    (SEQ ID NO:8)                     

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 2E1 contains oligonucleotides with thesequences

    (5') CTCCTCGTCATATCCATCTG (3')                                                                      (SEQ ID NO:9)                                              - (5') GCAGCCAATCAGAAATGTGG (3')     (SEQ ID NO:10).                   

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 3A1 contains oligonucleotides with thesequences

    (5') ATCCGATATGGAGATCAC (3')                                                                        (SEQ ID NO:11)                                             - (5') GAAGAAGTCCTTGTCTGC (3')       (SEQ ID NO:12).                   

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 3A2 contains oligonucleotides with thesequences

    (5') CGACTTGGAACCCATAGAC (3')                                                                       (SEQ ID NO:13)                                             - (5') GGCTTAGGGAGATTTGACATG (3')    (SEQ ID NO:14).                   

The primer set used for the amplification and detection of ratcytochrome P450 isoenzyme 4A1 contains oligonucleotides with thesequences

    (5') GGTGACAAAGAACTACAGC (3')                                                                       (SEQ ID NO:15)                                             - (5') AGAGGAGTCTTGACCTGCCAG (3')    (SEQ ID NO:16).                   

The primer set used for the amplification and detection of rat fattyacyl-CoA oxidase contains oligonucleotides with the sequences

    (5') CTGTTATGATGCTGCAGACAGC (3')                                                                    (SEQ ID NO:17)                                             - (5') ACACAGGTTCCTCAGCACAG (3')     (SEQ ID NO:18).                   

A primer set used for the amplification and detection of rat cyclophilincontains oligonucleotides with the sequences

    (5') CTTCGACATCACGGCTGATGG (3')                                                                     (SEQ ID NO:19)                                             - (5') CAGGACCTGTATGCTTCAGG (3')      (SEQ ID NO:20).                  

Another primer set used for the amplification and detection of ratcyclophilin contains oligonucleotides with the sequences

    (5') CTTGTCCATGGCAAATGCTG (3')                                                                      (SEQ ID NO:21)                                             - (5') GTGATCTTCTTGCTGGTCTTGC (3')   (SEQ ID NO:22).                   

The primer set used for the amplification and detection of rat CYP2B1contains oligonucleotides with the sequences

    (5') CAACCCTTGATGACCGCAGT (3')                                                                      (SEQ ID NO:23)                                             - (5') GGAAGTGTTCAGGATTGAAGC (3')    (SEQ ID NO:24).                   

The primer set used for the amplification and detection of ratUDP-Glucuronosyl-transferase contains oligonucleotides with thesequences

    (5') GCCTTCTCACTGCCTTGAAG (3')                                                                       (SEQ ID NO:25)                                            - (5') GTCTTACGGCAACAAAAGAGGCAG (3')  (SEQ ID NO:26).                  

The primer set used for the amplification and detection of ratGlutathione-S-transferase contains oligonucleotides with the sequences

    (5') CTTGGCAAAAGACAGGACC (3')                                                                       (SEQ ID NO:27)                                             - (5') GTTTTGCATCCATGGGAAGC (3')   (SEQ ID NO:28).                     

The primer set used for the amplification and detection of ratSulfotransferase contains oligonucleotides with the sequences

    (5') CTTCAGTTCCAAGGCCAAGG (3')                                                                      (SEQ ID NO:29)                                             - (5') GTGAAGTGATTCTTCCAGTC (3')     (SEQ ID NO:30).                   

Whereas most of the CYP450 enzymes have been shown to be constitutivelyexpressed in rat liver, many are markedly increased in expression uponexposure to various chemicals. For example, in adults male rats,expression of CYP2C11 is constitutively expressed and noninducible inthe liver whereas the expression of CYP450 1A1, 2B1, 2E1 and 4A1 are allselectively induced following exposure to various chemicals. Thisincrease in CYP450 enzyme expression can occur via a number ofmechanisms, including increased transcription, stabilization of mRNA andincreased protein synthesis.

The present invention provides a rapid, high throughput, screening assayfor the assessment of CYP450, especially in rat, which can be automatedfor routine toxicology applications. It is anticipated that theoligonucleotide primer sets can be present in a kit, alone or incombination with other elements, to screen any number of samples for thepresence of one of, or a series of, CYP450 isoenzymes. A primer set kitmay contain any one or more of the primer sets, in any combination, asneeded for toxicology applications.

Using the technique known as RT-PCR, changes in CYP450 mRNA expressionwere measured as a surrogate to changes in apoprotein expression andsubstrate-specific enzymatic activity in the liver of chemically exposedrats. To illustrate the validity of this approach, several prototypicalinducers of liver CYP450 enzymes were selected to determine thecorrelation between changes in mRNA, protein and enzymatic activity. Theinducers included β-naphthoflavone (BNF) as an inducer of the CYP1Asubfamily, phenobarbital (PB) as an inducer of the CYP2B subfamily,dexamethasone (DEX) as an inducer of the CYP3A subfamily and clofibrate(CLO) as an inducer of the CYP4A subfamily and fatty acyl-CoA oxidase.In addition, metyrapone (MET) was utilized to show the effects of anenzyme activity inhibitor on the expression of CYP450 isoenzymes.

As shown in FIGS. 3a-3w, all experiments showed correlation between theexpression of CYP450 enzymes at the mRNA and protein levels. Theseresults indicate that CYP450 enzyme expression in general is regulatedprimarily at the mRNA level. This is particularly evident in cases wherecertain of the compounds tested (e.g., BNF) were found to markedlyreduce CYP450 mRNA and apoprotein expression and enzyme activity.(Expression of the CYP2E1 isoenzyme, which is generally increased viaprotein stabilization, is an exception to this mechanism of CYP450enzyme regulation.) Additionally, for most of the CYP450 enzymes (i.e.,CYP450 1A1, 2B1/2, 3A1 and 4A1), expression of mRNA and apoproteincorrelated with enzymatic activity against selective substances. Minordifferences were identified only for the CYP2C11 and CYP2E1 isoenzymes,where the enzymatic substrates are known to be less specific.

Treatment of rats with BNF was found to cause increases in theexpression of both CYP1A1 and CYP1A2 mRNA, CYP1A1 apoprotein andcorresponding EROD activity. PB was also found to cause a slightincrease in both CYP1A1 apoprotein and EROD activity, but not in eitherCYP1A1 or CYP1A2 mRNA.

Treatment of rats with BNF was also found to cause a marked decrease inthe expression of the male specific CYP450 isoenzyme, 2C11. This effecthas been observed using a number of aromatic hydrocarbons and is relatedto an effect on the pituitary release of growth hormone and subsequentsuppression of androgen production, a phenomena often termed as the"feminization" of CYP450 expression. This effect was particularlyprominent at the mRNA expression level. Thus, the PT-PCR technique canbe used to assess the potential effects of xenobiotics on endocrinefunction.

PB treatment resulted in the prototypical increase in CYP2B and CYP3A1expression. CYP3A2 was not induced by PB in these studies; this effecthas been reported, Gonzales, et al., Mol. Cell. Bio. 6: 2969-2976, 1986.MET was also found to be an inducer of the CYP2B and CYP3A1 enzymes.These findings indicate that inhibitors of CYP450 enzyme activity haveno readily detectable effect on their capacity to cause enzyme inductionat either the mRNA or apoprotein levels.

Dexamethasone treatment caused a prototypical pattern of CYP450expression in rat liver, resulting in a selective increase in theexpression of CYP3A1. As in the case of PB, DEX did not induce theexpression of the CYP3A2 enzyme, demonstrating that these two isoenzymesof CYP450 are differentially regulated in the rat liver.

The hypolipidemic agent, CLO, was found to be a broad spectrum inducerof CYP450 in the rat liver, inducing an increase in the expression ofCYP450 3A1, 3A2, 4A1 and FACO using all three methods of detection.These findings demonstrate that in addition to peroxisome proliferatoractivity, CLO causes an increase in the expression of multiple CYP450enzymes. Moreover, CLO was found to consistently induce the expressionof CYP2E1 at both the mRNA and apoprotein levels in our studies.

mRNA expression as measured by RT-PCR can be used to accurately monitorthe changes in expression of most CYP450 isoenzymes in rat liverfollowing exposure to xenobiotics. Given the advantages of selectivity,sensitivity and speed of this versus traditional methods, these studiesdemonstrate the utility of RT-PCR for analysis of CYP450 expression inthe rat during routine toxicology studies.

EXAMPLE 1 (Application to an In Vivo Test System)

Male Sprague-Dawley rats between the ages of 6-8 weeks and weighing200-300 g were used in these studies. Food and water were available adlibitum.

Animals were administered either phenobarbital (PB) (100 mg/Kg),β-napthoflavone (BNF) (100 mg/Kg), isoniazid (INH) (200 mg/Kg,metyrapone (MET) (100 mg/Kg, dexamethasone (DEX) (200 mg/Kg) orclofibrate (CLO) (250 mg/Kg) for 4 consecutive days via intraperitoneal(i.p.) injection. Animals treated with water (H₂ O) and corn oil (CO)were used as controls. Two hours following the last injection (day 4),animals were sacrificed and the livers removed. The livers wereimmediately frozen and stored at -70° C.

Preparation of Total Liver RNA

Total RNA was prepared from frozen liver tissue using a modification ofthe method described by Xie and Rothblum, Biotechniques 11:326-327,1991. Approximately 100-200 mg. of liver tissue was homogenized in RNAextraction buffer to isolate total RNA. The resulting RNA wasreconstituted in diethylpyrocarbonate-treated water (DEPC-H₂ O),quantitated spectrophotometrically at 260 nm and adjusted to 100 μg/ml.Total RNA was stored in DEPC-H₂ O at -70° C. without any apparentdegradation.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

For conversion of total RNA to cDNA, a 20 μl reaction mix was preparedcontaining 1× reverse transcriptase (RT) buffer (GIBCO BRL), 10 nM DTT(dithiothreitol) 0.5 nM dTPs (deoxynucleotide triphosphates), 2.5 μMoligo d(T)₁₅ primer, 40 units RNasin, 200 units RNase H-RT (GIBCO BRL)and 400 ng of total RNA (in DEPC-H₂ O). The reaction was incubated for 1hour at 37° C. followed by inactivation of the enzyme at 95° C. for 5minutes. cDNA was stored at -20° C. until use.

For the PCR amplification of cDNA, a 10 μl reaction mix was preparedcontaining 10× polymerase reaction buffer, 2 mM MgCl₂, 1 unit Taq DNApolymerase, 20 ng cDNA and 200 nM of the 5' and 3' specific PCR primers.PCR reactions were carried out using melting, annealing and extensioncycling conditions of 94° C. for 30 seconds, 56° C. for 1 minute and 72°C. for 1 minute. All amplifications were carried out for 23 cycles.Under these conditions, all cDNA fragment amplifications were found toproduce single products within a linear range of 20-26 cycles. AmplifiedcDNA products were separated by PAGE (polyacrylamide gelelectrophoresis) using 5% native gels. Gels were stained with ethidiumbromide and photographed on a transilluminator using Polaroidpositive/negative film. The amount of each product was quantitateddensitometrically using a scanning laser densitometer.

Preparation of Liver Microsomes

Liver microsomes were prepared from frozen livers as described in Chenget al., J. Biol. Chem. 259: 12279-12284, 1984. Microsomes wereresuspended in buffer containing 10 mM Tris-HCl, 1 mM EDTA, and 20%glycerol. Microsomes were snap frozen in liquid nitrogen and stored at-80° C. until use. Total protein was measured using the bicinchonic acid(BCA) method using bovine serum albumin (BSA) as a standard.

SDS-PAGE and Western Immunoblotting

Proteins were separated on 10% SDS-PAGE gels under reducing conditionsand immunoblotted for detection of CYP450 isoenzymes using modificationsof previously reported methods, Harris, et al., P.N.A.S., U.S.A. 88:1407-1410, 1991. Proteins were loaded at 50 μg/lane and resolved underconstant current (250 V) for approximately 4 hr at 2° C. Proteins weretransferred to nitrocellulose membranes in 15 mM Tris buffer containing120 mM glycine and 20% (v/v) methanol. The nitrocellulose membranes wereblocked with 2.5% BSA and immunoblotted for CYP450 isoenzymes usingprimary monoclonal and polyclonal antibodies and secondary alkalinephosphatase conjugated anti-IgG. Immunoblots were developed with theBio-Rad alkaline phosphatase substrate kit.

Microsomal Enzyme Activity Measures

All microsomal protein samples were compared to positive controlmicrosomes with known enzymatic activities.

p-Nitrophenol Hydroxylation. The hydroxylation of p-nitrophenol wasmeasured spectrophotometrically as described by Koop, Chem. Res.Toxicol. 3: 377-383, 1990. The generation of 4-nitrocatechol wasdetermined from the absorbance at 546 nm as based on an extinctioncoefficient of 9.52 mM-¹ cm⁻¹. A DW2C dual-beam spectrophotometer wasused for these determinations.

7-Ethoxyresorufin O-Dealkylation (EROD) and 7-PentoxyresorufinO-Dealkylation (PROD). The O-dealkylation of 7-ethoxyresorufin and7-pentoxyresorufin was measured fluorometrically using the methodsdescribed by Burke, et al., Biochem. Pharmacol. 34: 3337-3345, 1985,with minor modifications, Dutton, et al., Arch. Biochem. Biophys. 268:617-629, 1989. The amount of resorufin generated was measuredfluorometrically (λ_(ex) ≈535 nm, λ_(ex) ≈585 nm).

Testosterone Oxidation. The oxidation of testosterone was determined byreverse-phase HPLC as described by Wood, et al. J. Biol. Chem. 258:8839-8847, 1983, Sonderfan, et al., Arch. Biochem. Biophys. 255: 27-41,1987, and Sonderfan, et al., Arch. Biochem. Biophys. 265: 208-218, 1988.Testosterone, androstenedione, 6-dehydrotestosterone and12-hydroxytestosterone isomers (i.e., 6α-, 15β-, 6β-, 15α-, 7α-, 16α-,16β-, 1β-, 18-, 11β-, 2α- and 2β-hydroxytestosterone) were resolved on aSupelcosil LC-18 octyldecylsilane (C₁₈) column and quantitated byintegration of peak areas.

Lauric Acid Hydroxylation. The rate of lauric acid hydroxylation wasdetermined by a combination of the radiometric partition method of Gieraand van Lier, Fund. Appl. Toxicol. 16: 348-355, 1991, and theradiometric HPLC method of Romano, et al., Anal. Biochem. 170: 83-93,1988. The rate of lauric acid hydroxylation was measured as the rate ofconversion of [¹⁴ C]-lauric acid to 11- and 12-hydroxylauric acid.Abbreviations: NCE, new chemical entities; ECM, extracellular matrix;DMEM, Dulbecco's Modified Eagel Medium; DMSO, dimethyl sulfoxide;CYP450, cytochrome P450; RT-PCR, reverse transcriptase-polymerase chainreaction; mRNA, messenger ribonucleic acid; PB, phenobarbital; HC,hydrocortisone-21-hemisuccinate; 3MC, 3-methylcholanthrene; CLO,clofibrate; FACO, fatty acyl-CoA oxidase; CYC, cyclophilin; UDPGT-PB,uridine diphosphate-glucuronosyltransferase (phenobarbital inducibleform); GST-Ya, glutathione-S-transferase-Ya; ST, rat sulfotransferase.

Results

The oligonucleotide PCR primers used to amplify rat CYP450 1A1, 1A2,2B1/2, 2C11, 2E1, 3A1, 3A2, 4A1, fatty acyl-CoA oxidase (FACO) andcyclophilin cDNA are as described above. The house-keeping gene,cyclophilin (Cavicchioli, et al., Mol. Brain Res. 9: 319-325, 1991), wasused to determine the constitutive level of gene transcription and tocontrol for variations in RNA recoveries from each liver specimen.Normalization was accomplished by quantitating the amount of amplifiedcDNA products by scanning laser densitometry and calculating the ratioof the amount of each enzyme cDNA relative to the amount of cyclophilin(CYC) cDNA (i.e., the enzyme cDNA O.D.:CYC cDNA O.D.). Thisenzyme:cyclophilin ratio was generated for each CYP450 isoenzyme andused to compare the relative amounts of CYP450 isoenzyme mRNA in eachliver.

Analysis of CYP1A1 and CYP1A2 Expression

The expression of CYP1A1 and CYP1A2 mRNA following exposure to variousinducers of CYP450 is presented in FIGS. 1 and 3a-3d. Under linearamplification conditions, expression of the CYP1A1 and CYP1A2 mRNA wasnot detected in the livers of water (H₂ O) and corn oil (CO) treatedcontrol rats. The expression of both genes was found to be noticeablyincreased following exposure of rats to the prototypical CYP1A inducer,β-naphthoflavone (BNF). One of 3 rats appeared to have a slight increasein CYP1A2 expression in response to dexamethasone (DEX) in thisexperiment. No other treatments were found to change the expression ofCYP1A1 or CYP1A2 at the mRNA level.

FIGS. 2 and 3c illustrate the detection of the CYP1A1 protein by Westernimmunoblotting of microsomal protein fractions isolated from theselivers. These results demonstrate close agreement in the detection ofCYP1A1 gene expression by the two techniques. A slight increase inCYP1A1 protein was observed in the livers of the phenobarbital (PB)treated rats (FIG. 3c). This increase in CYP1A1 expression was confirmedby an increase in microsomal EROD activity (FIG. 3d). CYP1A1 protein wasfound to be markedly increased after 3MC treatment.

Results of the analysis of microsomal EROD activity in the livers ofexposed rats confirmed the induction of CYP1A1 by BNF at both the mRNAand protein levels and the induction of CYP1A1 by PB at the proteinlevel (FIG. 3d). No other chemical treatments were found to cause anincrease in liver EROD activity over control levels.

Analysis of CYP2B1/2 Expression

The combined expression of CYP2B1 and CYP2B2 mRNA in male rat liverfollowing exposure to the various CYP450 inducers is presented in FIGS.1 and 3e-3h. These two enzymes have a 97% sequence homology and areco-regulated by chemical inducers of this CYP450 subfamily. Theoligonucleotide primers used in this study do not distinguish betweenthe two CYP2B isoenzymes.

CYP2B1/2 mRNA was found to be constitutively present in control (H₂ Oand CO treated) rat livers (FIGS. 1 and 3e). Following exposure of ratsto PB, a prototypical CYP2B inducer, expression of CYP2B1/2 mRNA wasfound to increase noticeably relative to control. Metyrapone (MET),hydrocortisone-21-hemisuccinate (HC) and clofibrate (CLO) were alsofound to cause an increase the expression of CYP2B1/2 mRNA relative totheir respective controls.

Similar results were obtained using Western immunoblotting forexpression of the CYP2B1 apoprotein in rat liver microsomal proteinfraction (FIG. 3f). The CYP2B1 apoprotein was found to be constitutivelyexpressed at a relatively low level in control rat livers and induciblyincreased following exposure to PB. Likewise, MET and CLO (to a lesserextent) were also found to cause an increase in the expression of theCYP2B1 apoprotein.

These findings were further confirmed by analysis of microsomal PROD andtestosterone 16-beta-hydroxylase activity in the livers of chemicallyexposed rats (FIGS. 3g and 3h). Increases in PROD and testosterone16-beta-hydroxylase activity were detected in both the PB and METtreated livers. CLO was not found to increase microsomal PROD activityin these livers. Induction of CYP2B at the mRNA and protein levels wasdetermined to be extremely weak and likely to be below the level ofdetection by enzymatic assay techniques.

Analysis of CYP2C11

High levels of mRNA expression of the adult male specific CYP450isoenzyme, CYP2C11, were detected in control (H₂ O and CO) rat livers(FIGS. 1 and 3i). Following exposure of rats to BNF (and MET to a lesserextent), a decreased expression of CYP2C11 mRNA was observed. None ofthe chemical treatments used in these studies were found to increaseCYP2C11 mRNA, confirming the high level constitutive expression of thisenzyme in the male rat.

Western immunoblot analysis of microsomal protein fractions for CYP2C11apoprotein expression also demonstrated the high level constitutiveexpression of this enzyme in control rats and a decreased expressionfollowing exposure of animals to BNF (FIGS. 3j and 3i). DEX was alsofound to have a slight effect on CYP2C11 protein. This effect of DEX wasnot observed at the mRNA level.

Analysis of microsomal testosterone 2-alpha-hydroxylase (T2aH) activityalso showed considerable CYP2C11 enzyme activity present in control ratlivers which was decreased following exposure to BNF (FIG. 3k). A slightdecrease was also observed in some of the rats exposed to DEX,confirming the decrease observed in apoprotein in these livers. Incontrast to both the mRNA and protein determinations, T2aH activity wasalso found to be decreased in the livers of animals exposed to PB.

Analysis of CYP2E1 Expression

Analysis of CYP2E1 mRNA demonstrated a high level constitutiveexpression in control male rat livers (FIG. 1 and 31). Followingexposure to the various prototypical CYP450 inducers, both HC and CLOwere found to increase CYP2E1 expression at the mRNA level. Moderateincreases in CYP2E1 apoprotein were also observed with HC and CLO and3MC. In some experiments CLO was found to cause a 2-3 fold increase inCYP2E1 mRNA expression. No other chemical treatments were found toaffect the expression of CYP2E over control levels.

As with mRNA, high levels of CYP2E1 apoprotein were found to beexpressed in control rat liver (FIGS. 2 and 3m). However, none of thecompound treatments, including CLO, were found to cause a consistenteffect on the levels of CYP2E1 apoprotein. BNF was found to cause aslight, but consistent, reduction in the levels of CYP2E1 apoprotein.

Two microsomal enzyme activities were monitored to assess the inductionof CYP2E1 activity, p-Nitrophenol hydroxylase (pNPH) and lauric acid11-hydroxylase (LA-11-H) (FIGS. 3n and 3o). Although they are eachindicative of CYP2E1 activity, neither is specific for the CYP2E1enzyme. The activity of pNPH was found to closely parallel the inductionof CYP2E1 protein, where PB and MET treatments both caused increases inenzyme activity. In addition, BNF was found to cause a marked reductionin pNPH activity. LA-11-H activity was also found to be increased by PBand decreased by BNF treatments but was not found to be increased by CLOexposure which reflects the ability of this compound to induce the CYP4Asubfamily of enzymes. pNPH activity has been demonstrated to moreaccurately assess CYP2E1 enzyme expression than LA-11-H.

Analysis of CYP3A1 and CYP3A2 Expression

CYP3A1 was found to be constitutively expressed in control male ratliver (FIGS. 1 and 3p-3s) whereas CYP3A2 was found to be almostundetectable in this cell culture system. Following exposure of rathepatocytes to HC, increase in both CYP3A1 and CYP3A2 mRNA levels wasobserved as compared to vehicle controls. PB was also found to cause anincrease in the expression of the CYP3A1 (and to much lesser extent,CYP3A2) mRNA levels relative to vehicle controls. Both HC and PB werealso found to increase CYP3A1 apoprotein expression as detected bywestern immunoblotting (FIGS. 2 and 3k). CLO was also found to causeslight elevations (two fold over control) in the expression of CYP3A1 atboth the mRNA and protein levels. Analysis of CYP3A2 protein expressionwas not determined in this study. 3MC did not cause an increase in theexpression of CYP3A1 or CYP3A2 at either the mRNA or protein level thanCYP3A1. Following exposure to PB, MET, DEX and CLO, CYP3A1 mRNA wasfound to be increased relative to control whereas CYP3A2 mRNA expressionwas only increased in CLO exposed livers.

Western immunoblot analysis of liver microsomal protein fractions forCYP3A1 apoprotein showed a similar profile of expression for this enzyme(FIG. 3r). As indicated by the expression of CYP3A1 mRNA, PB, MET andDEX exposures all resulted in marked increases in the apparent levels ofthe CYP3A1 apoprotein.

Analysis of liver testosterone 6-beta-hydroxylase (T6bH) activitysupports the changes in CYP3A mRNA and apoprotein expression (FIG. 3s).Although CLO was not found to cause an apparent increase in the levelsof liver CYP3A1 protein in this experiment, the induction of CYP3A1 mRNAwas confirmed by an increase in T6bH enzyme activity.

Analysis of CYP4A1 and FACO Expression

CYP4A1 and fatty acyl-CoA oxidase (FACO) mRNA were both found to beconstitutively expressed in control male rat livers (FIGS. 1, 3t and3w). Following exposure to CLO, a prototypical CYP4A1 inducer, bothCYP4A1 and FACO mRNA were increased. DEX caused only a slight increasein CYP4A1 mRNA expression (FIG. 1 and 3t) and was not found to affectthe expression of FACO.

A similar profile of CYP4A1 expression was found using Westernimmunoblot analysis of microsomal protein fractions were CLO was foundto cause a marked increase in the expression of the CYP4A1 apoprotein(FIG. 3u). DEX was not found to cause any increase in the CYP4A1apoprotein. Increased CYP4A1 expression by CLO was further demonstratedby an increase in lauric acid 12-hydroxylase (LA-12-H) activity in livermicrosomal protein fractions from exposed rats (FIG. 3v).

EXAMPLE 2 (Application Using an In Vitro Test System)

Male Sprague-Dawley rats between the ages of 6-8 weeks and weighing200-250 g were used in these studies. Food and water were available adlibitum.

Chemicals and Reagents

Cell culture materials. Cell culture medium was obtained from Gibco BRL(Grand Island, N.Y.). Collagenase type II was purchased from WorthingtonBiochemicals (Freehold, N.J.). Matrigel® was obtained from CollaborativeBiomedical Products (Bedford, Mass.). Percoll was obtained fromPharmacia Biotech (Uppsala, Sweden). Hydrocortisone-21-hemisuccinate,insulin, heat inactivated newborn bovine serum, bovine albumin and theantibiotic-antimycotic solution were obtained from Sigma (St. Louis,Mo.). All other chemicals were of the highest grade available andpurchased from Sigma. Tissue culture dishes (6 well plates) wereobtained from Costar (Cambridge, Mass.). RNA STAT⁶⁰ (total RNA isolationreagent) was purchased from Leedo Medical Laboratory (Houston, Tex.).

RT-PCR and Western Immunoblotting. The oligonucleotide PCR primers forthe amplification of rat CYC and CYP2B1 primers used here differ fromthose previously reported for the following reasons. The CYC primerspreviously used were designed to selectively amplify rat cyclophilincDNA. The primers according to the present invention are non-speciesselective and have been used to produce an equal length amplificationproduct of mouse, rat, dog, hamster, monkey and human cyclophilin. TheCYP2B primers previously used were non-selective for the rat cytochromeCYP2B enzymes and were unable to distinguish the CYP2B1 and CYP2B2enzyme cDNAs (e.g. CYP2B1/2 non-selective). The inventors' primers usedto amplify CYP2B have been designed to selectively amplify the ratCYP2B1 enzyme.

Cell Culture Medium Preparation: The culture medium used in this studywas specially formulated Dulbecco's Modified Eagel Medium (DMEM) witharginine free buffer, catalog #87-5128, as previously reported by Davilaet al. The stock culture medium (DMEM) was supplemented at all timeswith hydrocortisone (0.1 μM), insulin (1 μM) and sodium bicarbonate (2g/L). This serum free medium preparation was complemented with albuminand antibiotics and used to prepare the Matrigel® suspension asdescribed below.

Hepatocyte Isolation and Culturing

The procedure for the isolation and primary culture of parenchymalhepatocytes from rat liver was based on the two step collagenaseperfusion technique described by Seglen. Several modifications have beenmade to improve the culturing of the cell as indicated below.

The hepatocyte isolation procedure utilizes a retrograde perfusion ofthe liver via a catheter inserted through the right atrium and into theinferior vena cave. A peristaltic pump was used to perfuse the liverwith two buffer solutions: 1) a calcium and magnesium-free salt solution(BSS, pH 7.4) containing sodium chloride (8.3 g/L), potassium chloride(0.5 g/L) and HEPES (2.4 g/L) and 2) a dissociating buffer (pH 7.4)containing BSS and collagenase type II (100 U/mL). These solutions weremaintained at 37° C. and allowed to run as waste through a cut made inthe hepatic portal vein. A perfusion rate of 25 mL/min was maintainedfor both perfusates for about 10 minutes each.

After the perfusion was terminated, the liver was rapidly excised fromthe body cavity and transferred to a sterile beaker containing 200 mL ofwarm BSS. The liver was raked gently with a pair of blunt forceps. Thecell suspension was then filtered through a sterile nylon mesh (100 μm)and collected in several 50 ml conical test tubes. Hepatocytes werecentrifuged twice at 30× g for three minutes and then the final pelletwas resuspended with 30 mL of DMEM solution. Ten mL of diluted Percollsolution (40% v/v) was underlayed in each tube containing the cellsuspension and then centrifuged at 1100× g for 15 minutes. The pelletswere washed twice with DMEM at 30× g for 3 min and then resuspended in30 mL DMEM solution.

Viable and nonviable cells were counted using the trypan blue dyeexclusion test and a hemocytometer. Viability of the isolatedhepatocytes used in these studies was typically greater than 95%. Asolution of diluted Matrigel® (0.35 mg/mL DMEM) containing newbornbovine serum (8% v/v), albumin (0.1% w/v) and an antibiotics/antimycoticsolution (1% v/v) was prepared. The final cell suspension wasresuspended in this diluted Matrigel® solution to a final cell densityof 4×10⁵ cells/mL. A total of 8×10⁵ cells were plated per well (2 mLtotal volume) in 6 well culture plates.

The entire perfusion protocol from anesthesia to cell plating wascompleted within 2 hr. Hepatocytes were incubated for three days in ahumidified environment (95%) containing 5% CO₂. Hepatocytes werereplenished with fresh serum free-medium containing Matrigel® (0.35mg/mL), albumin (0.1% w/v) and the antibiotics/antimycotic solution(0.1% v/v) at 2 hr and 24 hr after plating. At 48 hr, the cultures werereplenished with plain DMEM medium without Matrigel®, albumin orantibiotic/antimycotic for the remainder of the cell culture period.

Cell Culture Treatments

Hepatocytes were treated with PB (100 μM), HC (20 μM), 3MC (1 μM), andCLO (100 μM). PB and HC were dissolved in DMEM whereas 3MC and CLO weredissolved in DMSO and then diluted with DMEM prior to administration tothe cultures (final DMSO concentration was 0.1% v/v). DMSO at 0.1% v/v,under the culture conditions described above, does not affect cellviability nor mRNA expression of the enzymes tested in this study (datanot shown). The concentration of inducers used in these studies wereselected based on preliminary experiments to determine the concentrationof inducer producing maximal levels of enzyme expression (data notshown).

Isolation of Total RNA

At the end of the treatment period, 72 hr after culture initiation,cultured hepatocytes (2 dishes per treatment) were rinsed with ice coldphosphate buffer saline (PBS) and harvested for total RNA or protein.Total RNA was extracted and measured as previously described using RNASTAT⁶⁰ reagent [33].

Microsomes and Total Protein preparation

Microsomes and total protein were prepared as described by Morris et al.using a total of 5 cultured plates (6 well plates) per treatment.

Semi-Quantitative RT-PCR and Western Immunoblotting

RT-PCR and western immunoblotting analysis of total RNA and microsomalprotein fractions for P450 enzyme expression were carried out aspreviously described. For the western immunoblotting of P450 enzymes,specific protein bands were detected using enhanced chemiluminescencereagent (DuPont NEN Research Product, Boston, Mass.). All studies wererepeated a minimum of five times and representative data are presented.

Results

Normalization of the data was accomplished by quantitating the amount ofamplified cDNA products by scanning laser densitometry (e.g. opticaldensity, O.D.) and calculating the ratio of the amount of each enzymecDNA relative to the amount of CYC cDNA. FIG. 1 illustrates the resultsobtained using RT-PCR to assess the levels of CYP450, FACO, UDPGT, ST,GST-Ya and CYC mRNA in rat hepatocytes exposed to various inducers ofCYP450. The results are presented in a series of graphs (FIG. 3) thatnormalize the results obtained for each gene product to that of thehousekeeping gene, CYC (i.e., enzyme O.D.:CYC O.D. ratio). Resultsobtained from western immunoblotting are compared to those curtainedwith RT-PCR for the detection of CYP450 expression (FIGS. 2 and 3).

The inventors confirmed that cell viability and composition of both theextracellular matrix and the culture medium are critical for themaintenance of multiple liver-specific functions in cultured rathepatocytes both at the mRNA and protein levels. The inventorsdetermined that the induction of hepatic CYP450, as well as Phase IIconjugating enzymes, by drugs and chemicals are markedly enhanced by thefollowing conditions: 1) high percent viability of isolated parenchymalcells (>94%), 2) adding insulin (1 μM) and hydrocortisone (100 nM) tothe serum-free culture medium, which at these concentrations, are noteffective inducing agents of the enzymes assessed in this study, 3)resuspending the hepatocytes directly into a diluted Matrigele solutionand then plating them in a non-extracellular matrix-coated culture dish;the optimum concentration of Matrigel® in these studies being found tobe 0.35 mg/mL, 4) replenishing the cells with fresh diluted Matrigel® ina serum-free medium at 2 hr and 24 hr after plating, and 5) adding thexenobiotics or CYP450 enzyme inducers at 24 and 48 hr after plating.Applicants found that a continuous 48 hr exposure to these enzymeinducing agents results in the induction of enzyme expression at boththe mRNA and protein levels. Under these culture conditions, hepatocytesremain responsive to enzyme inducers for a minimum of one week.

In concentration-ranging studies using 3MC (1.1 μM to 10 μM), PB (1 μMto 2 mM), HC (1 μM to 100 μM) and CLO (1 μM to 2 mM), it was establishedthat hepatocytes were responsive to chemical enzyme inducers in adose-related fashion as early as 24 hr after initial plating. Resultsfrom these studies were as follows: 1) 3MC increased expression ofCYP1A1 and CYP1A2 mRNAs at concentrations as low as 0.1 μM with anoptimum concentration at 1 μM, 2) PB caused a marked induction of theCYP2B1 enzyme mRNA at concentrations as low as 10 μM, with maximuminduction occurring at a concentration of 100 μM, 3) HC induced CYP3A1and CYP3A2 (to a much lesser degree) mRNAs expression at concentrationsas low as 10 μM, with a maximum induction occurring at a concentrationof 20 μM, and 4) CLO increased CYP4A1 at concentrations as low as 20 μM,with maximum induction occurring at a concentration of 100 μM. Theoptimum concentrations for these inducing agents were used for mRNA andprotein expression analysis in this study. Cell viability (>95%) wasmeasured at the beginning and at the end of the experiments using thetrypan blue dye exclusion test and cytosolic enzyme leakage assays,including lactate dehydrogenase (LDH), alanine aminotransferases (ALT)and sorbitol dehydrogenase (SDH).

Analysis of UDPGT, GST-Ya, and ST Expression

UDPGT, GST-Ya and ST mRNA was found to be constitutively expressed inrat hepatocytes cultured on Matrigel®, as indicated in FIGS. 1 and 3x-z.UDPGT was found to be markedly increased following exposure to PB and HCas compared to vehicle control cells. GST-Ya mRNA expression was foundto be increased following exposure to 3MC, PB, and CLO and ST mRNA wasinduced by HC. Analysis of UDPGT, GST-Ya and ST protein expression wasnot determined.

Although the present invention has been illustrated with reference tocertain preferred embodiments, it will be appreciated that the presentinvention is not limited to the specifics set forth therein. Thoseskilled in the art readily will appreciate numerous variations andmodifications within the spirit and scope of the present invention, andall such variations and modifications are intended to be covered by thepresent invention, which is defined by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 30                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTGGTTCTGG ATACCCAGCT G           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CCTAGGGTTG GTTACCAGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GTCACCTCAG GGAATGCTGT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GTTGACAATC TTCTCCTGAG G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GAGTTCTTCT CTGGGTTCCT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - ACTGTGGGTC ATGGAGAGCT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CTGCTGCTGC TGAAACACGT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGATGACAGC GATACTATCA C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CTCCTCGTCA TATCCATCTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GCAGCCAATC AGAAATGTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - ATCCGATATG GAGATCAC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GAAGAAGTCC TTGTCTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CGACTTGGAA CCCATAGAC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GGCTTAGGGA GATTTGACAT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GGTGACAAAG AACTACAGC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - AGAGGAGTCT TGACCTGCCA G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CTGTTATGAT GCTGCAGACA GC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - ACACAGGTTC CTCAGCACAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CTTCGACATC ACGGCTGATG G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CAGGACCTGT ATGCTTCAGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CTTGTCCATG GCAAATGCTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - GTGATCTTCT TGCTGGTCTT GC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - CAACCCTTGA TGACCGCAGT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - GGAAGTGTTC AGGATTGAAG C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - GCCTTCTCAC TGCCTTGAAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - GTCTTACGGC AACAAAAGAG GCAG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - CTTGGCAAAA GACAGGACC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - GTTTTGCATC CATGGGAAGC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - CTTCAGTTCC AAGGCCAAGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Rattus no - #rvegicus                                           (F) TISSUE TYPE: Liver                                                        (G) CELL TYPE: Hepatocy - #te                                        - -   (viii) POSITION IN GENOME:                                                       (C) UNITS: bp                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - GTGAAGTGAT TCTTCCAGTC            - #                  - #                      - # 20                                                                 __________________________________________________________________________

What is claimed is:
 1. A primer set for specifically detectingexpression of DNA encoding cytochrome P450 isoenzyme 1A1 in a tissuefollowing exposure to drugs or chemicals, the primer set comprising:oligonucleotides which consist essentially of CTGGTTCTGGATACCCAGCTG (SEQID NO:1) and CCTAGGGTTGGTTACCAGG (SEQ ID NO:2).
 2. A primer set forspecifically detecting expression of DNA encoding cytochrome P450isoenzyme 1A2 in a tissue following exposure to drugs or chemicals, theprimer set comprising: oligonucleotides which consist essentially ofGTCACCTCAGGGAATGCTGTG (SEQ ID NO:3) and GTTGACAATCTTCTCCTGAGG (SEQ IDNO:4).
 3. A primer set for specifically detecting expression of DNAencoding cytochrome P450 isoenzyme 2B1/2 in a tissue following exposureto drugs or chemicals, the primer set comprising: oligonucleotides whichconsist essentially of GAGTTCTTCTCTGGGTTCCTG (SEQ ID NO:5) andACTGTGGGTCATGGAGAGCTG (SEQ ID NO:6).
 4. A primer set for specificallydetecting expression of DNA of cytochrome P450 isoenzyme 2C11 in atissue following exposure to drugs or chemicals, the primer setcomprising: oligonucleotides which consist essentially ofCTGCTGCTGCTGAAACACGTG (SEQ ID NO: 7) and GGATGACAGCGATACTATCAC (SEQ IDNO:8).
 5. A primer set for specifically detecting expression of DNAencoding cytochrome P450 isoenzyme 2E1 in a tissue following exposure todrugs or chemicals, the primer set comprising: oligonucleotides whichconsist essentially of CTCCTCGTCATATCCATCTG (SEQ ID NO:9) andGCAGCCAATCAGAAATGTGG (SEQ ID NO:10).
 6. A primer set for specificallydetecting expression of DNA encoding cytochrome P450 isoenzyme 3A1 in atissue following exposure to drugs or chemicals, the primer setcomprising: oligonucleotides which consist essentially ofATCCGATATGGAGATCAC (SEQ ID NO:11) and GAAGAAGTCCTTGTCTGC (SEQ ID NO:12).7. A primer set for specifically detecting expression of DNA encodingcytochrome P450 isoenzyme 3A2 in a tissue following exposure to drugs orchemicals, the primer set comprising: oligonucleotides which consistessentially of CGACTTGGAACCCATAGAC (SEQ ID NO:13) andGGCTTAGGGAGATTTGACATG (SEQ ID NO:14).
 8. A primer set for specificallydetecting expression of DNA of cytochrome P450 isoenzyme 4A1 in a tissuefollowing exposure to drugs or chemicals, the primer set comprising:oligonucleotides which consist essentially of GGTGACAAAGAACTACAGC (SEQID NO:15) and AGAGGAGTCTTGACCTGCCAG (SEQ ID NO:16).
 9. A primer set forspecifically detecting expression of DNA of cytochrome P450 isoenzyme2B1 in a tissue following exposure to drugs or chemicals, the primer setcomprising: oligonucleotides which consist essentially ofCAACCCTTGATGACCGCAGT (SEQ ID NO:23) and GGAAGTGTTCAGGATTGAAGC (SEQ IDNO:24).
 10. A method for specifically detecting expression of DNAencoding a target enzyme selected from the group consisting ofcytochrome P450 isoenzymes 1A1, 1A2, 2B1, 2B1/2, 2C11, 2E1, 3A1, 3A2, or4A1 in a tissue following exposure to drugs or chemicals, the methodcomprising the steps of:a) providing a sample of RNA extracted from thetissue; b) converting the sample of RNA to cDNA using reversetranscriptase and oligo d(T)₁₅₋₁₈, primers; c) amplifying the cDNA usingpolymerase chain reaction and a primer set of any one of claims 1-8 and9 to provide amplified cDNA; and d) separating and detecting theamplified cDNA to detect expression of said target enzyme.
 11. Themethod of claim 10, wherein said RNA is extracted from rat liver.